Fully human antibodies against human 4-1bb

ABSTRACT

Fully human antibodies and antigen-binding portions thereof that bind to human 4-1BB and that allow binding of human 4-1BB to a human 4-1BB ligand. In one aspect, the antibody is an IgG4 antibody. Also provided is a method for treating a disease in a subject comprising administering a therapeutically effective amount of the antibody to said subject.

This application is a continuation of U.S. Ser. No. 15/174,268, filedJun. 6, 2016, which is a continuation of U.S. Ser. No. 14/211,438, filedMar. 14, 2014, now U.S. Pat. No. 9,382,328, which is a continuation ofU.S. Ser. No. 13/368,710, filed Feb. 8, 2012, now U.S. Pat. No.8,716,452, which is a continuation of U.S. Ser. No. 12/653,137, filedDec. 9, 2009, now U.S. Pat. No. 8,137,667, which is a continuation ofU.S. Ser. No. 11/903,106, filed Sep. 20, 2007, now U.S. Pat. No.7,659,384, which is a divisional of U.S. Ser. No. 10/961,567, filed Oct.8, 2004, now U.S. Pat. No. 7,288,638, which claims benefit of U.S.Provisional Application No. 60/510,193, filed Oct. 10, 2003, each ofwhich is incorporated by reference herein.

FIELD OF THE INVENTION

The invention is directed to fully human antibodies and, morespecifically, to fully human antibodies to human 4-1BB (CD137).

BACKGROUND OF THE INVENTION

An extensive body of evidence has unequivocally demonstrated that somedegree of immune response against cancer exists in humans and animals.In cancer patients, cellular components of the immune system are able torecognize antigens expressed by tumor cells, such as differentiation ofoncofetal antigens or mutated gene products (S. Rosenberg, Nature,411:380-4 (2001); P. van der Bruggen et al., Immunological Rev.,188:51-64 (2002)). A number of clinical studies have shown thattumor-infiltrating lymphocytes have favorable prognostic significance(E. Halapi, Med. Oncol., 15(4):203-11 (1998); Y. Naito et al., CancerRes., 58(16):3491-4 (1998); L. Zhang et al., N.E. J. Med., 348(3):203-13(2003)). Furthermore, treatment with immunomodulators, such as cytokinesor bacterial products, cancer vaccines, or adoptive immunotherapy hasled to tumor regression in a number of patients (S. Rosenberg, Cancer J.Sci. Am. 6(S):2 (2000); P. Bassi, Surg. Oncol., 11(1-2):77-83 (2002); S.Antonia et al., Current Opinion in Immunol., 16:130-6 (2004)). Despitethese responses, immunity against cancer frequently fails to effectivelyeliminate tumor cells. The causes for this failure can be grouped intothree major categories: (i) impaired tumor recognition by immune cells,either by variable expression of tumor antigens or reduced expression ofclass I major histocompatibility complex (MHC); (ii) immunosuppressivetumor microenvironment, as a result of secretion of inhibitory cytokinesby tumor cells (e.g., TGF-β); and (iii) poor tumor immunogenicity due tothe lack of expression of co-stimulatory molecules on tumor cells, whichresults in the inability of the tumor cells to effectively stimulateT-cells. Advances in our understanding of the requirements for tumorantigen recognition and immune effector function indicate that apotential strategy to enhance an anti-tumor immune response is toprovide co-stimulation through an auxiliary molecule. Tumorantigen-specific T-cells require costimulation to initiate and maintaineffector functions. Thus, therapies that target costimulatory moleculescan be applied to modulate and enhance the immune response to tumors.

The current model for T-cell activation postulates that naive T-cellsrequire two signals for full activation: (i) a signal provided throughthe binding of processed antigens presented to the T-cell receptor bymajor histocompatibility complex (MHC) class I molecules; and (ii) anadditional signal provided by the interaction of co-stimulatorymolecules on the surface of T-cells and their ligands on antigenpresenting cells. Recognition of an antigen by a naive T-cell isinsufficient in itself to trigger T-cell activation. Without aco-stimulatory signal, T-cells may be eliminated either by death or byinduction of anergy. Signaling through the CD28 costimulatory moleculeappears to be key for the initiation of T-cell responses. However, CD137(4-1BB) signaling has been shown to be primordial for the maintenanceand expansion of the immune response to antigens, as well as, for thegeneration of memory T-cells.

CD137 (4-1BB) is a member of the tumor necrosis receptor (TNF-R) genefamily, which includes proteins involved in regulation of cellproliferation, differentiation, and programmed cell death. CD137 is a 30kDa type I membrane glycoprotein expressed as a 55 kDa homodimer. Thereceptor was initially described in mice (B. Kwon et al., P.N.A.S. USA,86:1963-7 (1989)), and later identified in humans (M. Alderson et al.,Eur. J. Immunol., 24: 2219-27 (1994); Z. Zhou et al., Immunol. Lett.,45:67 (1995)) (See, also, Published PCT Applications WO95/07984 andWO96/29348, and U.S. Pat. No. 6,569,997, hereby incorporated byreference (See, SEQ ID NO:2.)). The human and murine forms of CD137 are60% identical at the amino acid level. Conserved sequences occur in thecytoplasmic domain, as well as 5 other regions of the molecule,indicating that these residues might be important for function of theCD137 molecule (Z. Zhou et al., Immunol. Lett., 45:67 (1995)).Expression of CD137 has been shown to be predominantly on cells oflymphoid lineage such as activated T-cells, activated Natural Killer(NK) cells, NKT-cells, CD4CD25 regulatory T-cells, and also on activatedthymocytes, and intraepithelial lymphocytes. In addition, CD137 has alsobeen shown to be expressed on cells of myeloid origin like dendriticcells, monocytes, neutrophils, and eosinophils. Even though CD137expression is mainly restricted to immune/inflammatory cells, there havebeen reports describing its expression on endothelial cells associatedwith a small number of tissues from inflammatory sites and tumors.

Functional activities of CD137 on T-cells have been amply characterized.Signaling through CD137 in the presence of suboptimal doses of anti-CD3has been demonstrated to induce T-cell proliferation and cytokinesynthesis (mainly IFN-γ), and to inhibit activated cell death. Theseeffects have been observed with both murine and human T-cells (W.Shuford et al., J. Exp. Med., 186(1):47-55 (1997); D. Vinay et al.,Semin. Immunol., 10(6):481-9 (1998); D. Laderach et al., Int. Immunol.,14(10):1155-67 (2002)). In both humans and mice, co-stimulation enhanceseffector functions, such as IFN-γ production and cytotoxicity, byaugmenting the numbers of antigen-specific and effector CD8+ T-cells. Inthe absence of anti-CD3 signaling, stimulation through CD137 does notalter T-cell function, indicating that CD137 is a co-stimulatorymolecule.

The physiological events observed following CD137 stimulation on T-cellsare mediated by NF-κB and PI3K/ERK1/2 signals with separatephysiological functions. NF-κB signals trigger expression of Bcl-_(XL),an anti-apopotic molecule, thus resulting in increased survival, whereasPI3K and ERK1/2 signals are specifically responsible for CD137-mediatedcell cycle progression (H. Lee et al., J. Immunol., 169(9):4882-8(2002)). The effect of CD137 activation on the inhibition ofactivation-induced cell death was shown in vitro by Hurtado et al. (J.Hurtado et al., J. Immunol., 158(6):2600-9 (1997)), and in an in vivosystem in which anti-CD137 monoclonal antibodies (mabs) were shown toproduce long-term survival of superantigen-activated CD8+ T-cells bypreventing clonal deletion (C. Takahashi et al., J. Immunol., 162:5037(1999)). Later, two reports demonstrated, under different experimentalconditions, that the CD137 signal regulated both clonal expansion andsurvival of CD8+ T-cells (D. Cooper et al., Eur. J. Immunol.,32(2):521-9 (2002); M. Maus et al., Nat. Biotechnol., 20:143 (2002)).Reduced apoptosis observed after co-stimulation correlated withincreased levels of Bcl-_(XL) in CD8+ T-cells, while Bcl-2 expressionremained unchanged. Up-regulation of the anti-apoptotic genes Bcl-_(xL)and bfl-1 via 4-1BB was shown to be mediated by NF-κB activation, sincePDTC, an NF-κB-specific blocker, inhibited 4-1BB-mediated up-regulationof Bcl-_(xL) (H. Lee et al., J. Immunol., 169(9):4882-8 (2002)). On theother hand, clonal expansion of activated T-cells appears to be mediatedby increased expression of cyclins D2, D3, and E, and down-regulation ofthe p27^(kip1) protein. This effect occurs in both an IL-2 dependent andindependent fashion (H. Lee et al., J. Immunol., 169(9):4882-8 (2002)).

Altogether, CD137 stimulation results in enhanced expansion, survival,and effector functions of newly primed CD8+ T-cells, acting, in part,directly on these cells. Both CD4+ and CD8+ T-cells have been shown torespond to CD137 stimulation, however, it appears that enhancement ofT-cell function is greater in CD8+ cells (W. Shuford et al., J. Exp.Med., 186(1):47-55 (1997); I. Gramaglia et al., Eur. J. Immunol.,30(2):392-402 (2000); C. Takahashi et al., J. Immunol., 162:5037(1999)). Based on the critical role of CD137 stimulation in CD8+ T-cellfunction and survival, manipulation of the CD137/CD137L system providesa plausible approach for the treatment of tumors and viral pathogens.

Recently, the constitutive expression of CD137 on freshly isolateddendritic cells (DCs) was demonstrated in mice (R. Wilcox et al., J.Immunol., 169(8):4230-6 (2002); T. Futagawa et al., Int. Immunol.,14(3):275-86 (2002)) and humans (S. Pauly et al., J. Leukoc. Biol.72(1):35-42 (2002)). These reports showed that stimulation of CD137 onDCs resulted in secretion of IL-6 and IL-12, and, more importantly, itenhanced DC ability to stimulate T-cell responses to alloantigens.Furthermore, Pan et al. demonstrated that CD137 signaling in DCsresulted in upregulation of MHC Class I and costimulatory molecules, andproduced an increased ability of DCs to infiltrate tumors (P. Pan etal., J. Immunol., 172(8):4779-89 (2004)). Therefore, CD137 costimulationon DCs appears to be a novel pathway for proliferation, maturation, andmigration of DCs.

Activated Natural Killer (NK) cells express CD137 following stimulationwith cytokines (I. Melero et al., Cell Immunol., 190(2):167-72 (1998);R. Wilcox et al., J. Immunol., 169(8):4230-6 (2002)). Several reportsdemonstrated that NK cells appear to be critical for the modulation ofthe antitumor immune response induced by agonistic CD137 antibodies ((I.Melero et al., Cell Immunol., 190(2):167-72 (1998); R. Miller et al., J.Immunol., 169(4):1792-800 (2002); R. Wilcox et al., J. Immunol.,169(8):4230-6 (2002)). Depletion of NK cells significantly reduces theantitumor activity of anti-CD137 mabs. Ligation of CD137 on NK cellsinduces proliferation and IFN-γ secretion, but does not affect theircytolytic activity. Notably, in vitro, CD137-stimulated NK cellspresented an immunoregulatory or “helper” activity for CD8+ cytolyticT-cells resulting in expansion of activated T-cells. Therefore, CD137signaling on NK cells may modulate innate immunity to tumors.

A paradoxical effect has been described for CD137 stimulation in thatagonistic CD137 antibodies can induce suppression of the humoralresponses to T-cell antigens in primates and mouse models (H. Hong etal., J. Immunother., 23(6):613-21 (2000); R. Mittler et al., J. Exp.Med., 190(10):1535-40 (1999)). Notably, CD137 agonistic antibodies wereshown to produce significant amelioration of the symptoms associatedwith antibody dependent autoimmune diseases such as systemic lupuserythematosus and experimental autoimmune encephalomyelitis (J. Foell etal., N.Y. Acad. Sci., 987:230-5 (2003); Y. Sun et al., Nat. Med.,8(12):1405-13 (2002)). Recently, Seo et al. demonstrated that, in amouse model of rheumatoid arthritis, treatment with an agonisticanti-CD137 antibody prevented the development of the disease, andremarkably, blocked disease progression (S. K. Seo et al., Nat. Med.10;1099-94 (2004)). The mechanism responsible for this effect has notbeen well defined, but in the model of rheumatoid arthritis it was shownthat treatment with a CD137 agonistic antibody resulted in the expansionof IFN-γ-producing CD11C-CD8+ T cells. IFN-γ in turn stimulateddendritic cells to produce indolamine-2,3-dioxygenase (IDO), whichexerts immuno-suppressive activities. It has also been postulated thatCD137 signaling on antigen-activated CD4+ T-cells results in inductionof IFN-γ secretion which activates macrophages. Activated macrophagescan in turn produce death signals for B cells. Continuous signalingthrough CD137 on CD4+ T-cells may subsequently induce activation-inducedcell death (AICD) of these CD4+ activated T-cells. Therefore, byeliminating antigen-activated T-cells and B cells, a reduced antibodyresponse is observed and, consequently, a dramatic reduction ofTh2-mediated inflammatory diseases is observed (B. Kwon et al., J.Immunol., 168(11):5483-90 (2002)). These studies suggest a role for theuse of agonistic CD137 antibodies for the treatment of inflammatory orautoimmune diseases, without inducing a general suppression of theimmune system.

The natural ligand for CD137, CD137 ligand (CD137L), a 34 kDaglycoprotein member of the TNF superfamily, is detected mainly onactivated antigen-presenting cells (APC), such as B cells, macrophages,dendritic cells, and also on murine B-cell lymphomas, activated T-cells,and human carcinoma lines of epithelial origin (R. Goodwin et al., Eur.J. Immunol., 23(10):2631-41 (1993); Z. Zhou et al., Immunol. Lett.,45:67 (1995); H. Salih et al., J. Immunol., 165(5):2903-10 (2000)).Human CD137L shares 36% homology with its murine counterpart (M.Alderson et al., Eur. J. Immunol., 24: 2219-27 (1994)).

In addition to delivering signals to CD137-expressing cells, binding ofCD137 to CD137L initiates a bidirectional signal resulting in functionaleffects on CD137L-expressing cells. Langstein et al. demonstrated thatbinding of CD137-Ig fusion protein to CD137L on activated monocytesinduced the production of IL-6, IL-8, and TNF-α, upregulated ICAM, andinhibited IL-10, resulting in increased adherence (J. Langstein et al.,J. Immunol., 160(5):2488-94 (1998)). In addition, proliferation ofmonocytes was demonstrated along with a higher rate of apoptosis (J.Langstein et al., J. Leukoc. Biol., 65(6):829-33 (1999)). Theseobservations were confirmed by the studies of Ju et al. (S. Ju et al.,Hybrid Hybridomics, 22(5):333-8 (2003)), which showed that a functionalanti-CD137L antibody induced a high rate of proliferation of peripheralblood monocytes. Blocking the ligand resulted in inhibition of T-cellactivation. In addition, soluble CD137L was found in the serum ofpatients with rheumatoid arthritis and hematological malignancies (H.Salih et al., J. Immunol., 167(7):4059-66 (2001)). Thus, the interactionof CD137 with CD137L influences and produces functional effects onT-cells and APC.

In another important aspect of T-cell function, it has been demonstratedthat agonistic anti-CD137 antibodies rescued T-cell responses to proteinantigens in aged mice. It has been well documented that an age-relateddecline in the immune response to antigens occurs, a process known asimmunosenescence (R. Miller, Science, 273:70-4 (1996); R. Miller,Vaccine, 18:1654-60 (2000); F. Hakim et al., Curr. Opinion Immunol.,16:151-156 (2004)). This phenomenon appears to be due to alterations inthe equilibrium between the extent of cellular expansion and cellularsurvival or death. Bansal-Pakala et al. tested the hypothesis thatsecondary costimulation through CD137 can be used to enhance T-cellresponses in situations where T-cells do not receive sufficientstimulation, due to either reduced expression of CD3 or CD28, or reducedquality of signals. Their studies showed that aged mice had a deficientin vitro recall response compared to young mice (P. Bansal-Pakala etal., J. Immunol., 169(9):5005-9 (2002)). However, when aged mice weretreated with anti-CD137 mabs, the proliferative and cytokine responsesof T-cells were identical to the responses observed in young mice. Whilethe specific mechanism responsible for this effect was not elucidated,it was speculated that enhancing both the expression of anti-apoptoticmolecules like Bcl-_(XL), and the promotion of IL-2 secretion in vivomay play a role in rescuing defective T-cell responses. These studiesdemonstrated the potential for agonistic anti-CD137 antibodies to rescueweak T-cell responses in elderly immuno-compromised individuals, and hasprofound implications for the use of anti-CD137 antibodies in cancerpatients.

A role for CD137 targeted therapy in the treatment of cancer wassuggested by in vivo efficacy studies in mice utilizing agonisticanti-murine CD137 monoclonal antibodies. In a paper by Melero et al.,agonistic anti-mouse CD137 antibody produced cures in P815 mastocytomatumors, and in the low immunogenic tumor model Ag104 (I. Melero et al.,Nat. Med., 3(6):682-5 (1997)). The anti-tumor effect required both CD4+and CD8+ T-cells and NK cells, since selective in vivo depletion of eachsubpopulation resulted in the reduction or complete loss of theanti-tumor effect. It was also demonstrated that a minimal induction ofan immune response was necessary for anti-CD137 therapy to be effective.Several investigators have used anti-CD137 antibodies to demonstrate theviability of this approach for cancer therapy (J. Kim et al., CancerRes., 61(5):2031-7 (2001); O. Martinet et al., Gene Ther., 9(12):786-92(2002); R. Miller et al., J. Immunol., 169(4):1792-800 (2002); R. Wilcoxet al., Cancer Res., 62(15):4413-8 (2002)).

In support of the anti-tumor efficacy data with agonistic CD137antibodies, signals provided by CD137L have been shown to elicit CTLactivity and anti-tumor responses (M. DeBenedette et al., J. Immunol.,163(9):4833-41 (1999); B. Guinn et al., J. Immunol., 162(8):5003-10(1999)). Several reports demonstrated that gene transfer of CD137 ligandinto murine carcinomas resulted in tumor rejection, demonstrating therequirement of costimulation in generating an efficient immune response(S. Mogi et al., Immunology, 101(4):541-7 (2000); I. Melero et al., CellImmunol., 190(2):167-72 (1998); B. Guinn et al., J. Immunol.,162(8):5003-10 (1999)). Salih et al. reported the expression of CD137Lin human carcinomas and human carcinoma cell lines (H. Salih et al., J.Immunol., 165(5):2903-10 (2000)), and demonstrated that tumors cellsexpressing the ligand were able to deliver a co-stimulatory signal toT-cells which resulted in the release of IFN-γ and IL-2, and that thiseffect correlated with the levels of CD137L on tumors. Whetherexpression of CD137L in human tumors could make these tumors moresusceptible to agonistic CD137 antibodies is not known.

CD137L −/− mice have underscored the importance of the CD137/CD137Lsystem in T-cell responses to both viruses and tumors (M. DeBenedette etal., J. Immunol., 163(9):4833-41 (1999); J. Tan et al., J. Immunol.,164(5):2320-5 (2000); B. Kwon et al.,

J. Immunol., 168(11):5483-90 (2002)). Studies using CD137- andCD137L-deficient mice have demonstrated the importance of CD137costimulation in graft-vs-host disease, and anti-viral cytolytic T-cellresponses. CD137-deficient mice had an enhanced proliferation ofT-cells, but a reduction in cytokine production and cytotoxic T-cellactivity (B. Kwon et al., J. Immunol., 168(11):5483-90 (2002); D. Vinayet al., Immunol. Cell Biol., 81(3):176-84 (2003)). More recently, it wasshown that knockout mice (CD137−/−) had a higher frequency of tumormetastases (4-fold) compared to control mice. These data suggest thatrestoration of CD137 signaling by the use of agonistic anti-CD137antibodies is a feasible approach for augmenting cellular immuneresponses to viral pathogens and cancers.

In addition to the data in mouse in vivo models which supports theinvolvement of CD137 signaling in antitumor immune responses, studiesconducted in primary human tumor samples have confirmed the role ofCD137 in generating effector T-cells. In patients with Ewing sarcoma,Zhang et al. showed that intratumoral effector T-cells presented theCD3+/CD8+/CD28−/CD137+ phenotype. Unexpectedly, coexistence ofprogressive tumor growth and anti-tumor immunity (effector T-cells) wasobserved. Ex vivo stimulation studies with patients' cells demonstratedthat tumor-induced T-cell proliferation and activation requiredcostimulation with CD137L. Stimulation of PBL with anti-CD3/CD137L, butnot anti-CD³/anti-CD28, induced tumor lytic effectors. These studiesprovided further evidence that CD137 mediated costimulation could resultin expansion of tumor reactive CTLs (H. Zhang et al., Cancer Biol.Ther., 2(5):579-86 (2003)). Furthermore, expression of CD137 wasdemonstrated in tumor infiltrating lymphocytes in hepatocellularcarcinomas (HCC) (Y. Wan et al., World J. Gastroenterol, 10(2):195-9(2004)). CD137 expression was detected in 19 out of 19 HCC by RT-PCR,and in 13/19 by immunofluorescence staining. Conversely, CD137 was notdetected in the peripheral mononuclear cells of the same patients.Analyses conducted in healthy donor liver tissues failed to demonstrateexpression of CD137. These studies did not attempt to correlate clinicaldisease with CD137 expression. Thus, studies conducted in Ewing sarcomaand hepatocellular carcinoma revealed the presence of TIL that expressCD137, with concomitant disease progression. In Ewing sarcomas it wasdemonstrated that CD137+ TILs were able to kill tumor cells ex-vivosuggesting that the CD137 pathway was intact in these patients, and thatperhaps suppressive factors in the tumor microenvironment inhibitedtheir function. Hence, it can be postulated that systemic administrationof agonistic CD137 antibodies may provide the signal necessary forexpansion of these effector T-cells.

In addition to its role in the development of immunity to cancer,experimental data supports the use of CD137 agonistic antibodies for thetreatment of autoimmune and viral diseases (B. Kwon et al., Exp. Mol.Med., 35(1):8-16 (2003); H. Salih et al., J. Immunol., 167(7):4059-66(2001); E. Kwon et al., P.N.A.S. USA, 96:15074-79 (1999); J. Foell etal., N.Y. Acad. Sci., 987:230-5 (2003); Y. Sun et al., Nat. Med.,8(12):1405-13 (2002) S. K. Seo et al, Nat. Med. 10;1099-94 (2004)).

Consequently, based on the roles of 4-1BB in modulating immune response,it would be desirable to produce anti-human 4-1BB antibodies withagonistic activities that could be used for the treatment or preventionof human diseases such as cancer, infectious diseases, and autoimmunediseases.

BRIEF SUMMARY OF THE INVENTION

The present invention provides fully human antibodies that bind to human4-1BB (H4-1BB) and that allow binding of H4-1BB to a human 4-1BB ligand(H4-1BBL). Thus, the invention is directed to antibodies that bind toH4-1BB and that do not block the binding of H4-1BB to H4-1BBL, therebypermitting the binding of both an antibody of the invention and H4-1BBLto H4-1BB. The invention also provides antibodies with agonisticactivities in that binding of the antibodies to H4-1BB results in anenhancement and stimulation of H4-1BB mediated immune responses. Theseantibodies can be used as immuno-enhancers of an anti-tumor oranti-viral immune response, or as immunomodulators of T cell mediatedautoimmune diseases. The antibodies can also be used as diagnostic toolsfor the detection of H4-1BB in blood or tissues of patients with cancer,autoimmune, or other diseases.

In one aspect, the invention provides a monoclonal antibody orantigen-binding portion thereof that specifically binds to H4-1BB,comprising a light chain variable region and a heavy chain variableregion, wherein the light chain variable region comprises a CDR1(complementary determining region 1), a CDR2 (complementary determiningregion 2), and a CDR3 (complementary determining region 3) as depictedin FIG. 4, and the heavy chain variable region comprises a CDR1(complementary determining region 1), a CDR2 (complementary determiningregion 2), and a CDR3 (complementary determining region 3) as depictedin FIG. 3 or FIG. 7. The monoclonal antibody (mab) can be, for example,an IgG4 antibody or IgG1 antibody.

In another aspect, the invention provides a monoclonal antibody orantigen-binding portion thereof, wherein the light chain comprises avariable region as depicted in FIG. 4, and the heavy chain comprises avariable region as depicted in FIG. 3 or FIG. 7.

In another aspect, the invention provides a monoclonal antibodycomprising a light chain and a heavy chain, wherein the light chaincomprises amino acid residues 21-236 of SEQ ID NO:6 and the heavy chaincomprises amino acid residues 20-467 of SEQ ID NO:3. In another aspect,the invention provides a monoclonal antibody comprising a light chainand a heavy chain, wherein the light chain comprises amino acid residues21-236 of SEQ ID NO:6 and the heavy chain comprises amino acid residues20-470 of SEQ ID NO:9.

The antibodies of the invention have wide therapeutic applications asimmunomodulators of diseases such as cancer, autoimmune diseases,inflammatory diseases, and infectious diseases.

The invention further provides methods for treating cancer in a subjectcomprising administering a therapeutically effective amount of anantibody of the invention to the subject. In one aspect, this methodfurther comprises administering a vaccine. Suitable vaccines include,for example, a tumor cell vaccine, a DNA vaccine, a GM-CSF-modifiedtumor cell vaccine, or an antigen-loaded dendritic cell vaccine. Thecancer can be, for example, prostate cancer, melanoma, or epithelialcancer.

In another aspect, the invention provides a method for enhancing theimmune response, comprising administration of an antibody of theinvention and a SIV gag vaccine. In another aspect, the inventionprovides a method for enhancing the immune response, comprisingadministration of an antibody of the invention and a PSA vaccine. Inanother aspect, the invention provides a method for enhancing the immuneresponse to a SIV gag vaccine, comprising administration of an antibodyof the invention. In another aspect, the invention provides a method forenhancing the immune response to a PSA vaccine, comprisingadministration of an antibody of the invention.

The invention also provides pharmaceutical compositions comprising anantibody of the invention, or an antigen-binding portion thereof, and apharmaceutically acceptable carrier. The pharmaceutical composition canbe administered alone or in combination with an agent, e.g., an agentfor treating cancer such as a chemotherapeutic agent or a vaccine orother immunomodulatory agent.

The invention also provides isolated polynucleotides comprising anucleotide sequence selected from: (a) nucleotides that encode the aminoacid sequence of amino acid residues 20-467 of SEQ ID NO:3; (b)nucleotides that encode the amino acid sequence of SEQ ID NO:3; (c)nucleotides that encode the amino acid sequence of amino acid residues21-236 of SEQ ID NO:6; (d) nucleotides that encode the amino acidsequence of SEQ ID NO:6; (e) nucleotides that encode the amino acidsequence of amino acid residues 20-470 of SEQ ID NO:9; (f) nucleotidesthat encode the amino acid sequence of SEQ ID NO:9; and (g) nucleotidesthat encode a fragment of an amino acid sequence of (a) to (f), such asa variable region, constant region, or one or more CDRs. The isolatedpolynucleotides of the invention further comprise nucleotide sequencesencoding at least one CDR of FIG. 3, at least one CDR of FIG. 4, or atleast one CDR of FIG. 7. The invention further provides isolatedpolynucleotides that comprise the nucleotide sequence of SEQ ID NO:1,SEQ ID NO:4, or SEQ ID NO:7.

The invention also provides isolated polypeptides comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:6, and SEQ ID NO:9. In another aspect, the invention providesisolated polypeptides comprising the amino acid sequence of amino acidresidues 20-467 of SEQ ID NO:3, isolated polypeptides comprising theamino acid sequence of amino acid residues 21-236 of SEQ ID NO:6, andisolated polypeptides comprising the amino acid sequence of amino acidresidues 20-470 of SEQ ID NO:9. In another aspect, the inventionprovides isolated polypeptides comprising the amino acid sequence of atleast one CDR of FIG. 3, FIG. 4, or FIG. 7, or at least the variable orconstant region of FIG. 3, FIG. 4, or FIG. 7.

The invention further includes an immunoglobulin having bindingspecificity for H4-1BB, said immunoglobulin comprising an antigenbinding region. In one aspect, the immunoglobulin is a Fab or F(ab′)2 ofan antibody of the invention.

The invention also includes a cell line that produces an antibody orantigen-binding portion thereof of the invention, recombinant expressionvectors that include the nucleotides of the invention, and methods tomake the antibodies of the invention by culturing an antibody-producingcell line.

BRIEF DESCRIPTION OF THE DRAWINGS:

FIG. 1 shows a plasmid map of pD17-20H4.9.h4a.

FIG. 2 shows a plasmid map of pD16gate-20H4.9.LC.

FIGS. 3A-3H shows the nucleotide sequence of the plasmidpD17-20H4.9.h4a, including the coding strand (SEQ ID NO:1),complementary strand (SEQ ID NO:2), and amino acid sequence (leaderpeptide is amino acid residues 1-19 of SEQ ID NO:3; heavy chain is aminoacid residues 20-467 of SEQ ID NO:3) encoded by the coding strand.

FIGS. 4A-4F shows the nucleotide sequence of the plasmidpD16gate-20H4.9.LC, including the coding strand (SEQ ID NO:4),complementary strand (SEQ ID NO:5), and amino acid sequence (leaderpeptide is amino acid residues 1-20 of SEQ ID NO:6; light chain is aminoacid residues 21-236 of SEQ ID NO:6) encoded by the coding strand.

FIG. 5 shows a schematic of the 20H4.9-IgG1 heavy chain sequenceconstruct.

FIG. 6 shows a schematic of the 20H4.9 light chain sequence construct.

FIGS. 7A-7D shows the nucleotide and amino acid sequences of the20H4.9-IgG1 heavy chain construct, including the coding strand (SEQ IDNO:7), complementary strand (SEQ ID NO:8), and amino acid sequence(leader peptide is amino acid residues 1-19 of SEQ ID NO:9; heavy chainis amino acid residues 20-470 of SEQ ID NO:9) encoded by the codingstrand.

FIGS. 8A-8B illustrates the results obtained from the binding of mab20H4.9-IgG1 to human CD137 by ELISA (FIG. 8A) and the effect of mab20H4.9-IgG1 on CD137-CD137L interaction (FIG. 8B).

FIGS. 9A-9B illustrates the results obtained from the binding of mab20H4.9-IgG1 to PMA-ionomycin stimulated human or cynomolgus monkeycells. Human CEM (FIG. 9A) or monkey PBMC (FIG. 9B) were incubated with20H4.9-IgG1 or human CD137L fusion protein.

FIGS. 10A-10B illustrates the results obtained by induction of IFN-γ inco-stimulatory studies with anti-CD137 antibodies, which are expressedas fold increase in pg/ml over controls. Due to the variable backgroundresponse among donors, data was normalized relative to controltreatments (=1). Median IFN-γ baseline level for human T-cells (FIG.10A) or monkey PBMC (FIG. 10B) stimulated with anti-CD3 alone was 592pg/ml and 505 pg/ml respectively.

FIG. 11 provides plasmon resonance plots of binding of mab 20H4.9-IgG4and mab 20H4.9-IgG1 to human CD137.

FIG. 12 illustrates the concentration-dependent binding of 20H4.9-IgG4to PMA ionomycin stimulated human CEM cells, but no binding tounstimulated CEM cells. FIGS. 13A-13B illustrates the induction ofIFN-yin co-stimulatory studies with anti-CD137 antibodies. The resultsare expressed as fold increase in pg/ml over controls. Due to thevariable background response among donors, data was normalized relativeto control treatments (=1). Median IFN-γ baseline level for humanT-cells (FIG. 13A) or monkey PBMC (FIG. 13B) stimulated with anti-CD3alone was 592 pg/ml and 505 pg/ml respectively.

FIGS. 14A-14B illustrates the results obtained of dose-dependentenhancement of IFN-γ synthesis by mab 20H4.9-IgG4 (FIG. 14A), and effectof antibody crosslinking by addition of crosslinking anti-human IgGantibody (7 μg/ml) (FIG. 14B).

FIG. 15 illustrates the effect of mab 20H4.9-IgG4 on T-cell survival andcell cycle progression. Human T-cells were costimulated with anti-CD3 (1ug/ml)±mab 20H4.9-IgG4 at the concentrations listed. Six days afterinitiation of the assays, cells were collected and stained withAnnexin-V and propidium iodide to determine the number of live cells(Annexin V/PI negative), or PE-conjugated cyclin D2 to detect cyclingcells. Results represent the mean (±SD) of 4 lots of mab 20H4.9-IgG4tested in parallel.

FIGS. 16A-16D shows in cynomolgus monkeys the antigen-specific IFN-γresponse as measured by ELISPOT after treatment with a DNAvaccine±anti-human 4-1BB antibodies. Animals were treated with a SIV gagvaccine (day 0, 28, 56; FIG. 16A), SIV gag vaccine (day 0, 28, 56) andmab 20H4.9-IgG4 (day 12, 15 and 19; FIG. 16B), or SIV gag vaccine (day0, 28, 56) and hu39E3.G4 (day 12, 15 and 19; FIG. 16C). A group ofanimals was left untreated (FIG. 16D). At various times followingtreatment, blood was collected, and PBMC were separated and evaluatedfor their ability to secrete IFN-γ in the presence of antigenstimulation.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to the preparation and characterization ofantibodies, and antigen binding fragments thereof (including fusionproteins that comprise an antigen binding fragment of an antibody of theinvention), for use in the treatment of a disease, such as a cancer,infectious disease, inflammatory disease, or autoimmune disease. Thecancer can be, for example, prostate cancer, melanoma, or epithelialcancer.

The antibodies are capable of binding to H4-1BB, and can present highaffinity for H4-1BB and effectively enhance T cell responses. In oneaspect, the antibody induces IFN-γ production in co-stimulatory assays,but does not affect the binding of H4-1BB to its corresponding ligand,H4-1BBL, and does not fix complement.

The antibodies of the invention may be produced by methods well known inthe art. In one aspect, the antibodies can be produced by expression intransfected cells, such as immortalized eukaryotic cells, such asmyeloma or hybridoma cells.

The antibodies of the invention may be used alone, or together withother therapeutic agents such as radiotherapy (including radiation),hormonal therapy, cytotoxic agents, vaccines, and other immunomodulatoryagents, such us cytokines and biological response modifiers. Theseagents are particularly useful in treating cancer andimmune-proliferative disorders.

In one aspect, the invention provides the monoclonal antibody (mab)20H4.9-IgG4. FIGS. 1 and 2 provide plasmid maps of pD17-20H4.9.h4a andpD16gate-20H4.9.LC, respectively, that can be used to produce mab20H4.9-IgG4. FIG. 3 (FIGS. 3A-3H) provides the nucleotide sequence ofthe plasmid pD17-20H4.9.h4a, including the coding strand (SEQ ID NO:1),complementary strand (SEQ ID NO:2), and amino acid sequence (leaderpeptide is amino acid residues 1-19 of SEQ ID NO:3; heavy chain is aminoacid residues 20-467 of SEQ ID NO:3) encoded by the coding strand. FIG.4 (FIGS. 4A-4F) shows the nucleotide sequence of the plasmidpD16gate-20H4.9.LC, including the coding strand (SEQ ID NO:4),complementary strand (SEQ ID NO:5), and amino acid sequence (leaderpeptide is amino acid residues 1-20 of SEQ ID NO:6; light chain is aminoacid residues 21-236 of SEQ ID NO:6) encoded by the coding strand.

In another aspect, the invention provides the monoclonal antibody (mab)20H4.9-IgG1. FIG. 5 schematically shows a heavy chain sequence constructof mab 20H4.9-IgG1. FIG. 6 schematically shows a light chain sequenceconstruct of mab 20H4.9, for both mab 20H4.9-IgG4 and 20 H4.9-IgG1. FIG.7 provides the nucleotide sequence (coding strand (SEQ ID NO:7) andcomplementary strand (SEQ ID NO:8)) of the heavy chain sequenceconstruct of FIG. 5, and the amino acid sequence (leader peptide isamino acid residues 1-19 of SEQ ID NO:9; heavy chain is amino acidresidues 20-470 of SEQ ID NO:9) encoded by the coding strand. The lightchain of mab 20H4.9-IgG1 is the same as the light chain of mab20H4.9-IgG4.

The invention also encompasses antibodies with conservative amino acidsubstitutions from the heavy and light chain amino acid sequencesdepicted in SEQ ID NOS:3, 6, and 9 that have substantially no effect onH4-1BB binding. Conservative substitutions typically include thesubstitution of one amino acid for another with similar characteristics,e.g., substitutions within the following groups: valine, glycine;glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamicacid; asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine.

The polynucleotides encoding the polypeptides of the invention typicallyfurther comprise an expression control sequence operably linked to thepolypeptide coding sequences, including naturally-associated orheterologous promoter regions. Preferably, the expression controlsequences will be eukaryotic promoter systems in vectors capable oftransforming or transfecting eukaryotic host cells, but controlsequences for prokaryotic hosts may also be used. Once the vector hasbeen incorporated into an appropriate host, the host is maintained underconditions suitable for high level expression of the nucleotidesequences and, as desired, the collection and purification of the lightchain, heavy chain, light/heavy chain dimers or intact antibody, bindingfragments or other immunoglobulin form may follow. (See, S. Beychok,Cells of Immunoglobulin Synthesis, Academic Press, N.Y. (1979)). Singlechain antibodies or minibodies (single chain antibodies fused to one ormore CH domains) may also be produced by joining nucleic acid sequencesencoding the VL and VH regions disclosed herein with DNA encoding apolypeptide linker.

Prokaryotic hosts, such as E. coli, and other microbes, such as yeast,may be used to express an antibody of the invention. In addition tomicroorganisms, mammalian tissue cell culture may also be used toexpress and produce the antibodies of the invention. Eukaryotic cellsmay be preferred, because a number of suitable host cell lines capableof secreting intact immunoglobulins have been developed including, forexample, CHO (chinese hamster ovary) cell lines, COS (African greenmonkey fibroblast cell line) cell lines, HeLa cells, myeloma cell lines,and hybridomas. Expression vectors for these cells can includeexpression control sequences, such as a promoter or enhancer, andnecessary processing information sites, such as ribosome binding sites,RNA splice sites, polyadenylation sites, and transcriptional terminatorsequences, all well known in the art.

The vectors containing the DNA segments of interest (e.g., the heavyand/or light chain encoding sequences and expression control sequences)can be transferred into the host cell by well-known methods, which varydepending on the type of cellular host. For example, calcium chloridetransfection is commonly used for prokaryotic cells, whereas calciumphosphate treatment, lipofection, or electroporation may be used forother cellular hosts. (See, e.g., T. Maniatis et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Press (1982)).

Once expressed, the antibodies, their dimers, individual light and heavychains, or other immunoglobulin forms, can be purified according tostandard procedures in the art, such as ammonium sulfate precipitation,affinity columns, column chromatography, gel electrophoresis, and thelike. Substantially pure immunoglobulins of at least 90 to 95%homogeneity are desirable, and 98 to 99% or more homogeneity are moredesirable.

The antibodies of the invention are useful for modulating T cell andantibody-mediated immune responses. Typical disease states suitable fortreatment include cancers, infectious diseases, inflammatory diseases,and autoimmune diseases such as multiple sclerosis, rheumatoidarthritis, systemic lupus erythematosus, and myaesthenia gravis.

The invention also provides pharmaceutical compositions comprising atleast one antibody of the invention and a pharmaceutically acceptablecarrier. The pharmaceutical compositions may be sterilized byconventional well known sterilization techniques. The pharmaceuticalcompositions can also contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, stability enhancing agents such asmannitol or tween 80, toxicity adjusting agents and the like, forexample, sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate, or human albumin.

The antibodies and pharmaceutical compositions of the invention areparticularly useful for parenteral administration, includingsubcutaneous, intramuscular, and intravenous administration. Thepharmaceutical compositions for parenteral administration can comprise asolution of the antibody dissolved in an acceptable carrier, preferablyan aqueous carrier. A variety of aqueous carriers can be used, all wellknown in the art, e.g., water, buffered water, saline, glycine and thelike. These solutions are sterile and generally free of particulatematter. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage.

The pharmaceutical composition can further comprise an additional agentfor treatment of a disease. In one aspect, the pharmaceuticalcomposition includes an agent for treatment of a cancer, an infectiousdisease, inflammatory disease, or autoimmune disease. The antibody ofthe invention can also be co-administered or separately administeredwith an additional agent for treatment of a disease.

The antibodies of the invention can be used with other agents to enhancethe immune response to cancerous cells in a patient. In one aspect, theantibody is used in combination with an immunogenic agent, such ascancerous cells, purified tumor antigens (including recombinantproteins, peptides, and carbohydrate molecules), or cells transfectedwith genes encoding immune stimulating cytokines and cell surfaceantigens. In another aspect, the antibody is used in combination with avaccine such as, for example, a tumor cell vaccine, a DNA vaccine, agene-modified tumor cell vaccine, such as GM-CSF-modified tumor cellvaccine, a peptide vaccine, or an antigen-loaded dendritic cell vaccine.

Many experimental strategies for vaccination against tumors have beendevised. In one of these strategies, a vaccine is prepared usingautologous or allogeneic tumor cells. These cellular vaccines have beenshown to be most effective when the tumor cells are transduced toexpress GM-CSF. GM-CSF has been shown to be a potent activator ofantigen presentation for tumor vaccination (Dranoff et al., P.N.A.S.,90:3539-43 (1993); E. Jafee et al., J. Clin. Oncol., 19:145-56 (2001);R. Salgia et al., J. Clin. Oncol., 21:624-30 (2003)).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (S. Rosenberg, Immunity 10:281-7 (1999)). In many cases, thesetumor specific antigens are differentiation antigens expressed in thetumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp 100, MAGE antigens, Trp-2. Many of these antigenscan be shown to be the targets of tumor specific T cells found in thehost. The antibodies of the invention may be used in conjunction with acollection of recombinant proteins and/or peptides expressed in a tumorin order to amplify and direct the immune response to these antigenstowards a Thl response. These proteins are normally viewed by the immunesystem as self antigens and are therefore tolerant to them.

In one aspect of the invention, the antibody is combined with animmunodulatory agent comprising the SIV gag antigen (as a model for HIVDNA vaccine) or prostate specific antigen (PSA), or a DNA vaccinecomprising a nucleotide sequence that encodes the SIV gag antigen orprostate specific antigen (PSA). PSA vaccines are described in, forexample, M. Pavlenko et al., Br. J. Cancer, 91(4):688-94 (2004); J.Wolchok et al., Semin. Oncol., 30(5):659-66 (2003); J. Kim et al., Clin.Cancer Res., 7(3 Suppl.):882s-889s (2001). SIV gag vaccines aredescribed in, for example, B. Makitalo et al., J. Gen. Virol., 85(Pt8):2407-19 (2004); N. Letvin et al., J. Virol., 78(14):7490-7 (2004); S.Mossman et al., AIDS Res. Hum. Retroviruses., 20(4):425-34 (2004); F.Bertley et al., J. Immunol., 172(6):3745-57 (2004); L. Patterson et al.,J. Virol., 78(5):2212-21 (2004); E. O'Neill et al., AIDS Res. Hum.Retroviruses, 19(10):883-90 (2003); Z. Bu et al., Virology,309(2):272-81 (2003).

The tumor antigen may also include, for example, the protein telomerase,which is required for the synthesis of telomeres of chromosomes andwhich is expressed in more than 85% of human cancers and in only alimited number of somatic tissues (N. Kim et al., Science, 266,2011-2013 (1994)). Tumor antigen may also be “neo-antigens” expressed incancer cells because of somatic mutations that alter protein sequence orcreate fusion proteins between two unrelated sequences, or idiotype fromB cell tumors. Other tumor vaccines may include the proteins fromviruses implicated in human cancers such a Human Papilloma Viruses(HPV), Hepatitis Viruses (HBV and HCV), and Kaposi's Herpes SarcomaVirus (KHSV). Another form of tumor specific antigen which may be usedwith an antibody of the invention is purified heat shock proteins (HSP)isolated from the tumor tissue itself. These heat shock proteins containfragments of proteins from the tumor cells and these HSPs are highlyefficient at delivery to antigen presenting cells for eliciting tumorimmunity (R. Suot et al., Science 269: 1585-1588 (1995); Y. Tamura etal., Science 278: 117-120 (1997)).

The antibodies of the invention can also be used to enhance the immuneresponse to vaccines to viral antigens, such as HIV or HCV. Theantibodies of the invention can also be used to enhance the immuneresponse to other immunomodulatory agents, and to elicit a memory immuneresponse. Examples of these agents are cytokines such as GM-CSF, IL-2,IL-15, IL-12, F13 ligand, CD40 ligand, adjuvants such asCpG-oligodeoxynucleotide (bacterial DNA), or antibodies to OX-40 orCTLA-4.

The pharmaceutical compositions of the invention can be administered forprophylactic and/or therapeutic treatments. In therapeutic application,the pharmaceutical composition is administered to a patient alreadysuffering from a disease, in an amount sufficient to cure or at leastpartially arrest or treat the disease. An amount adequate to accomplishthis is defined as a “therapeutically effective dose.” Amounts effectivefor this use will depend upon the severity of the disease state and thepatient (including, for example, the general state of the patient's ownimmune system), and can be determined by one skilled in the art. Inprophylactic applications, the pharmaceutical composition isadministered to a patient not already in the disease state, to enhancethe patient's resistance to the disease state. Such an amount is definedto be a “prophylactically effective dose.” In this use, the preciseamounts depend upon the patient's state of health (including, forexample, the general state of the patient's own immune system), and canbe determined by one skilled in the art. In one aspect, the prophylacticuse is for the prevention of tumor recurrence.

EXAMPLES Example 1 Generation of Antibodies Materials and Methods

Fully human monoclonal antibodies to the human CD137 (4-1BB) receptorwere generated in the HuMAb-Mouse® (Medarex, Inc., Princeton, N.J.).HuMAb mice were immunized five times intraperitoneally (i.p.) andsubcutaneously (s.c.) with 25 μg of the extracellular domain of humanCD137 in RIBI adjuvant (Ribi Immunochemical). Prior to fusion, mice wereboosted intravenously (i.v.) with the same amount of antigen. Spleencells from immunized mice with adequate titers of antibodies to huCD137were fused to mouse myeloma cells following standard procedures.

Anti-human CD3 mab (clone:HIT3a), ELISA kits for human and monkey IFN-γ,cytometric bead array (CBA) kits, and all conjugated antibodies for flowcytometry were purchased from BD Pharmingen (San Diego, Calif.). HumanIgG₁ λ and Human IgG₁ κ were purchased from Sigma-Aldrich (St. Louis,Mo.). CEM cells (ATCC-CRL 2265) were purchased from ATCC. Culture media(RPMI), and fetal bovine serum (FBS) were purchased from Mediatech Inc.(Herndon, Va.). Sheep Red Blood Alsevers was purchased from ColoradoSerum Co. (Denver, Colo.).

Hybridoma screening: Detection of binding to huCD137 by ELISA: Toidentify hybridomas secreting anti-human CD137 antibodies, ELISA plates(Nunc MaxiSorp) were coated with human CD137-mouse IgG_(2b) fusionprotein at 1 μg/ml in PBS overnight at 4° C. Plates were then washed 3times with PBS with 0.01% Tween-80 (PBS-T), and subsequently blockedwith PBS-T plus 1% bovine serum albumin (BSA), for 20 min at roomtemperature. Fifty microliters of supernatants diluted 1:3 in PBS-T wereadded to the plates and incubated for 1-2 hr at ambient temperature.Afterwards, plates were washed as before, and binding of antibodies wasdetected by an incubation with alkaline phosphatase-conjugated goatF(ab′)2 anti-human IgG antibody (Jackson Laboratories, West Grove, Pa.).Plates were developed with pNPP and read at 405 nm.

Blocking assay: Twenty-six hybridomas secreting antibodies thatrecognized huCD137 by ELISA were evaluated for their ability to allowCD137-CD137L interactions. These analyses were conducted initially in anELISA format. Plates were coated with human CD137-muIgG_(2b) at 0.2μg/ml, 100 μl/well. Serial dilutions of the mab 20H4.9-IgG1, or controlantibodies, diluted in PBS-T and 1% bovine serum albumin, were added tothe plate. CD137L-CD8 fusion protein was added to the wells at aconcentration of 0.2 μg/ml. Binding of antibodies was detected with abiotinylated anti-CD8 antibody (0.2 μg/ml, Ancell Corporation, Bayport,Minn.). After several washes, streptavidin-alkaline phosphatase (1:2000)and pNPP for the detection of bound antibodies were added, and theplates were read at 405 nm.

To confirm that the selected antibodies did not alter CD137-CD137Lbinding, purified antibodies were further characterized by BlAcoreanalyses. All experiments were carried out on a BIAcore 3000 instrument(BlAcore Inc., Piscataway, N.J.). Human CD137 was immobilized covalentlyat a high density on a carboxy-methylated dextran surface of a BIAcoresensorchip (BlAcore Inc., Piscataway, N.J.). Injections were conductedat 2 μg/mL in 10 mM acetate buffer, pH 5.0. Unoccupied active esterswere subsequently blocked by injection of an excess of ethanolamine.Regeneration of the surface was done with 10 mM glycine, pH 2.0.

Purified samples of anti-CD137 antibodies were diluted to concentrationsbetween 200 and 1000 nM using HEPES buffered saline, pH 7.4,supplemented with 0.15 M NaCl and 0.005% surfactant P20 (HBS-EP). HumanCD137L-CD8 fusion proteins (huCD137L) were used as source of CD137ligand. Experiments were conducted in which huCD137L was injected priorto anti-CD137 antibodies, or vice versa. Injections were performed at aflow rate of 5 μL/min. Bound ligand and antibodies were removed byregeneration with 10 mM glycine buffer, pH 2.0.

Human T-cell purification: T-cells or PBMCs were obtained from healthyhuman donors. Blood was collected in EDTA, suspended in elutriationbuffer (RPMI containing 2.5 mM EDTA, 10 μg/ml polymyxin B), underlayedwith Lymphocyte Separation Medium (LSM, Mediatech Inc., Herndon, Va.),and centrifuged at 1800 rpm for 25 minutes. Cellular interfaces werecollected, and centrifuged at 1500 rpm for 10 minutes. Afterwards, cellpellets were resuspended in elutriation buffer and washed Sheep RedBlood Cells (SRBC, 1:10 dilution), and incubated on ice for 1 hour.Cells were then underlayed with LSM and centrifuged at 2500 rpm for 25minutes. Interfaces were removed and SRBC were lysed with SRBC LysisBuffer. Isolated T-cells were washed and resuspended in 10% FBS/RPMI.

Flow Cytometric analyses: Binding of anti-human CD137 antibodies toCD137 expressed on cells was determined by flow cytometry. A humanT-cell leukemia cell line (CEM) or cynomolgus monkey peripheral bloodmonocytic cells (PBMC) were used for these studies. These cells do notexpress CD137 constitutively, but the receptor can be induced bystimulation with phorbol myristate (PMA, 10 ng/ml) and ionomycin (1 μM)for 18 hr. Cells were then washed and incubated with variousconcentrations of the antibodies in staining buffer (phosphate buffersaline, PBS, plus 1% FCS, and 0.01% sodium azide). Binding of theantibodies to stimulated or non-stimulated cells was detected by afluorescein (FITC) or phycoerithrin (PE) conjugated goat anti-human IgG(Jackson Immunoresearch, West Grove, Pa.). To confirm expression ofCD137, a fusion protein consisting of the extracellular domain of CD137ligand and mouse CD8 was used (Ancell Corporation, Bayport, Minn.),followed by incubation with PE-conjugated anti-mouse CD8 (BD Pharmingen,San Diego, Calif.). Samples were fixed in 1% formalin, kept at 4° C.,and read by flow cytometry.

Functional assays: Primary human T-cells or monkey PBMC obtained fromhealthy donors were stimulated with immobilized anti-CD3 antibody toprovide the first signal for T-cell activation, and co-stimulated withhuman anti-human CD137 antibodies. As a non-specific control, ahumanized anti-carcinoma antibody (BR96) was used at the same antibodyconcentration. Plates were coated with anti-CD3 antibody (0.5-1 μg/ml)at 4° C. overnight. The next day T-cells or PBMC were plated at1-1.5×10⁵/well concentrations. Synthesis of IFN-γ was measured after 72hours of culture at 37° C. either by cytometric bead array (CBA) or byELISA.

Cytokine Assays

ELISA: After stimulation of T-cells at various times, plates werecentrifuged and media was removed. Cytokine levels were detected by anELISA in accordance with the manufacturer's instructions (BD Pharmingen,San Diego, Calif.). In brief, test samples and standards were added toanti-cytokine-coated 96-well plates. After incubation for 2 hr atambient temperature, plates were washed 3 times in PBS-T and thenincubated first with a working detector antibody, followed by theaddition of substrate. Absorbance was read at 405 nm, and concentrationswere calculated based on the standard curve.

Cytometric Bead Array: Another method used to determine cytokineproduction in vitro was flow cytometry using the Cytometric Bead array(CBA) developed by BD Pharmingen. Levels of IFN-γ, IL-2, IL-5, IL-4,IL-10, and TNF-α were measured in culture supernatants followingmanufacturers' instructions. Results were analyzed by flow cytometrywith the CBA analysis software.

Results

Hybridomas secreting antibodies that showed binding to human CD137 werefurther expanded, and subcloned. Secreted antibodies were purified andtested for their ability to bind to huCD137 and to allow the interactionof CD137-CD137L. Of the panel of anti-human CD137 antibodies evaluated,mab 20H4.9-IgG1 was selected for further evaluation based on its bindingprofile and non-blocking properties. The 20H4.9-IgG1 antibody is IgG1kappa as determined by ELISA using alkaline phosphatase anti-human IgG1,2, 3, 4, and anti-kappa and lambda reagents (Southern Biotech,Birmingham, Alabama). FIG. 8 (FIG. 8A—binding to human CD137 by ELISA;FIG. 8B—effect of mab 20H4.9-IgG1 on CD137-CD137L interaction) providesthe initial characterization of mab 20H4.9-IgG1. Serial dilutions of mab20H4.9-IgG1, 26G6 (a blocking anti-CD137 antibody), or tetanus toxoid(TT, negative control) were evaluated for their ability to alter bindingof CD137 to CD137L. Mab 20H4.9-IgG1 at concentrations up to 10 μg/ml didnot block CD137L binding, whereas mab 26G6 inhibited binding atconcentrations >0.37 μg/ml.

Mab 20H4.9-IgG1 was also tested for reactivity towards CD137 expressedon human T-cells (CEM) and in cynomolgus monkey peripheral bloodmonocytic cells (PBMC) stimulated with PMA and ionomycin. Previousstudies determined that CD137 is upregulated on T-cells followingactivation with PMA and ionomycin. Control molecules consisted of anirrelevant human IgG antibody (negative control) or CD137L-CD8 fusionprotein (positive control, BD Pharmingen, San Diego, Calif.). Resultsfrom these studies indicated that mab 20H4.9-IgG1 bound to activatedhuman CEM and PBMCs from cynomolgus monkeys, with minimum binding tounstimulated cells. Similar percentages of positive cells were detectedwith either mab 20H4.9-IgG1 or CD137L. FIG. 9 provides the resultsobtained demonstrating the binding of mab 20H4.9-IgG1 to PMA-ionomycinstimulated human or cynomolgus monkey cells. Human CEM (FIG. 9A) ormonkey PBMC (FIG. 9B) were incubated with 20H4.9-IgG1 or human CD137Lfusion protein. Secondary antibodies were added and samples were read byflow cytometry.

Next, it was determined whether mab 20H4.9-IgG1 could induce enhancementof IFN-γ in costimulatory assays in the presence of anti-CD3stimulation, the key functional effect desired for an agonistic CD137antibody. Mab 20H4.9-IgG1 was evaluated for its co-stimulatory activityin functional studies in human and monkey lymphocytes. Based on initialdata, a concentration of 20 ug/ml anti-CD137 antibody (excess antibody)was used in these studies. Levels of anti-CD3 antibody between 0.2-1μg/ml were tested which resulted in 10-20% CD137-positive lymphocytes.Levels of IFN-γ in supernatants were measured after 72 h of culture. Asshown in FIG. 10, mab 20H4.9-IgG1 enhanced IFN-γ synthesis in both humanand monkey costimulatory assays to levels significantly higher thancontrols. Results of studies conducted with T-cells isolated from 8healthy human donors showed that in six of them, mab 20H4.9-IgG1enhanced IFN-γ synthesis between 2.2-4.3-fold compared to controls. Oneof the other two donors showed a 1.6-fold increase. The level ofenhancement was superior to that observed with hu39E3.G4, a humanizedanti-CD137 antibody provided in published PCT Application WO04/010947(herein incorporated by reference) which showed augmentation of IFN-γ in5 out of 8 donors and at levels lower than mab 20H4.9-IgG1 (1.5-2-foldincrease) (FIG. 10A). In monkey costimulatory studies, mab 20H4.9-IgG1also demonstrated enhanced functional activity resulting in significantaugmentation of IFN-γ over controls (FIG. 10B). As in the human studies,enhancement of IFN-γ was consistently higher than with hu39E3.G4.

Induction of TNF-α synthesis above control levels was also observed inhuman cultures, albeit at much lower levels than IFN-γ. TNF-α levelsinduced by anti-CD3 antibody alone (baseline) were about 20-50 foldlower than baseline levels for IFN-γ. Mab 20H4.9-IgG1 induced anincrease of ˜2 to 4.7-fold in 3 out of 8 donors. Again, hu39E3.G4(tested in parallel) induced ˜2-fold increase in the same donors but atlower levels. Other cytokines tested, IL-2, IL-5, IL-10, and IL-4 didnot change significantly with either treatment.

Together these studies demonstrated that mab 20H4.9-IgG1 presented thefunctional activity desired in both humans and monkeys by inducing aTh1-type of response. Significantly, since in vivo anti-tumor activityis associated with the ability of anti-CD137 antibodies to induce IFN-γsynthesis, these results supported the selection of mab 20H4.9-IgG1 forisotype switching.

Example 2 In Vitro Characterization of Mab 20H4.9-IgG4

Based on its binding kinetics, inability to block CD137-CD137Linteraction, and functional effects on human T-cells, mab 20H4.9-IgG1was selected for switching to an IgG4 form. The IgG4 form of mab20H4.9-IgG1 is 20H4.9-IgG4 (depicted in FIGS. 3 and 4).

The second phase of these studies involved the comparison of the invitro properties of mab 20H4.9-IgG4 and mab 20H4.9-IgG1. In thissection, the binding kinetic properties, and functional effects of bothantibodies in human and monkey lymphocytes are described.

Binding Kinetics

Kinetic properties of anti-human CD137 antibodies were evaluated bysurface plasmon resonance using a BlAcore 3000 instrument. The antigen,human CD137-murine IgG2a, was immobilized covalently at a low density onthe surface of a CMS sensorchip. Mab 20H4.9-IgG4 and mab 20H4.9-IgG1were injected at concentrations between 25 and 200 nM. FIG. 11 depictsinjections at 100 nM for both mab 20H4.9-IgG1 and mab 20H4.9-IgG4. Datacalculated using BlAevaluation software (bivalent model, global curvefit analysis) resulted in kinetic parameters that were similar for bothantibodies (see Table 1). Dissociation constants KD for mab 20H4.9-IgG1and mab 20H4.9-IgG4 were determined as 11.2 and 16.6 nM, respectively.Under similar experimental conditions, mab 20H4.9-IgG4 did not bind tomurine 4-1BB.

TABLE 1 Comparison of the binding kinetics of mab 20H4.9-IgG4 and mab20H4.9-IgG1 ka2 K_(D)1 antibody k_(a1) (1/Ms) k_(d1) (1/s) (1/RUs) kd2(1/s) Rmax (RU) K_(A)1 (nM) 20H4.9- 3.43E+04 3.85E−04 2.30E−05 1.51E−03262 8.91E+07 11.22 IgG1 20H4.9- 3.92E+04 6.51E−04 0.0755 0.105 4096.02E+07 16.61 IgG4

Flow Cytometric Analyses

Biotinylated mab 20H4.9-IgG4 at concentrations ranging from 0.32 ng/mlto 5 μg/ml was tested for binding to CEM cells±PMA-ionomycin. Mab20H4.9-IgG4 bound to PMA-ionomycin stimulated CEM cells in aconcentration-dependent manner. Saturation was achieved at 0.2 μg/ml. Onthe other hand, as shown for its parental molecule, mab 20H4.9-IgG1, mab20H4.9-IgG4 did not bind to CEM cells that were not stimulated withPMA-ionomycin (FIG. 12). Concentration-dependent binding of mab20H4-.9-IgG4 was demonstrated in PMA-ionomycin stimulated CEM cells(FIG. 12). Samples were read by flow cytometry.

Cellular/Functional Assays

To confirm that the process of switching the isotype of mab 20H4.9-IgG1did not alter the activity of the antibody, in vitro studies wereconducted to compare the activity of mab 20H4.9-IgG4 to the parent mab20H4.9-IgG1 in monkey PBMC and human T-cells. The functional effects ofmab 20H4.9-IgG4 on human and monkey T-cells or PBMC were determined andcompared to its parental molecule, mab 20H4.9-IgG1. Primary humanT-cells or monkey PBMC obtained from healthy donors were stimulated withanti-CD3 antibody (0.5 μg/ml-1 μg/ml) +/− anti-human CD137 antibodies.Synthesis of IFN-γ was measured after 72 h of culture at 37° C. bycytometric bead array (CBA) for human samples or by ELISA for monkeysamples. Antibodies were tested in costimulatory assays in the presenceof suboptimal concentrations of anti-CD3 antibody (1 μg/ml) orConcavalin A (1 μg/ml) (donors M5170 and 81 only). Results are expressedas fold increase in pg/ml over controls. Due to the variable backgroundresponse among donors, data was normalized relative to controltreatments (=1). FIG. 13A provides the human T-cell results and FIG. 13Bprovides the monkey PBMC results. As shown in FIGS. 13A-13B, mab20H4.9-IgG4 demonstrated costimulatory properties yielding higher levelsof IFN-γ in human and monkey cells compared to controls. The level ofenhancement of IFN-γ synthesis was comparable to its parental moleculein human and monkey samples.

Subsequently, the effect of antibody cross-linking on the functionaleffect of mab 20H4.9-IgG4 was evaluated. It has been shown thatcross-linking of antibodies may result in potentiation of theirsignaling ability. Thus, a study was conducted to determine thefunctional activity of several batches of mab 20H4.9-IgG4 ±an anti-humanIgG antibody. As shown in FIG. 14A, significant enhancement of IFN-γsynthesis was observed for all lots tested in the absence ofcross-linking antibodies, with a plateau at concentrations of 400 ng/ml.The augmentation of IFNy synthesis by mab 20H4.9-IgG4 was furtherenhanced by the addition of anti-human IgG cross-linking antibody asshown in FIG. 14B. Different batches of mab 20H4.9-IgG4 had comparablecellular activities.

Thus, cross-linking of mab 20H4.9-IgG4 resulted in an enhancement of theability of the antibody to induce IFN-γ synthesis. Antibodycross-linking in vivo may occur by cellular receptors for the Fc portionof immunoglobulins or by antibody dimerization. Mab 20H4.9-IgG4 is ofthe IgG4 isotype, which, compared to other IgG isotypes, has lowaffinity for Fc receptors. However, IgG4 can bind to FcyRI (CD64)expressed on monocytes and neutrophils.

Two other approaches were used to further characterize mab 20H4.9-IgG4:(i) effect on T-cell survival and (ii) effect on cyclin D2 expression.To determine whether mab 20H4.9-IgG4 could elicit signaling throughCD137 on human T-cells and provide co-stimulatory signals to T-cellsleading to cell survival and expansion, human T-cells stimulated withanti-CD3 antibodies +/− mab 20H4.9-IgG4 at concentrations known toinduce IFN-γ synthesis were stained with annexin-V and propidium iodideto determine the number of live cells (Annexin V/Propidium iodidenegative), and with Cyclin D2 to determine its effect on cellprogression. FIG. 15 shows the average results of 4 different lots ofmab 20H4.9-IgG4 on cyclin D2 expression and survival of T-cells.

Concentrations of mab 20H4.9-IgG4 of 0.4-10 μg/ml resulted in anincrease in the number of live cells by approximately 1.8-2 fold, andyielded a significant increase in the number of cyclin D2-expressingT-cells (2.5-3 fold).

Example 3 In Vivo Evaluation of 4-1BB Antibodies in a PharmacodynamicModel in Cynomolgus Monkeys

This example illustrates the ability of mab 20H4.9-IgG4 and mabhu39E3.G4 to enhance the antigen specific immune response elicited byDNA vaccines. Materials and Methods

Experimental animal groups: Female and male cynomolgus monkeys (2.5 to5.0 kg) were purchased from Charles River BRF (Houston, Tex.) for thisstudy and were housed in pairs. Each experimental group consisted of 4males and 2 females which were randomized into groups by body weight.Experimental groups were as follows:

-   -   Group 1—SIV gag and PSA DNA vaccine (2 mg each), day 0, 28, 56,        i.m., plus saline control, i.v., on days 12, 15 and 19;    -   Group 2—SIV gag and PSA DNA vaccine (2 mg each), day 0, 28, 56,        i.m., plus mab hu39E3.G4, i.v., on days 12, 15 and 19;    -   Group 3 - SIV gag and PSA DNA vaccine (2 mg each), day 0, 28,        56, i.m., plus mab 20H4.9-IgG4, i.v., on days 12, 15 and 19;    -   Group 4—untreated control group.

Immunizations and antibody treatments: PSA and SIV gag DNA vaccines wereobtained from David B. Weiner, Department of Pathology and Laboratory ofMedicine, University of Pennsylvania. (See, Kim et al., Oncogene 20,4497-4506 (2001); Muthumani et al., Vaccine 21, 629-637 (2003).)

Monkeys were immunized by the intramuscular route with both PSA and SIVgag DNA constructs (2 mg/construct/immunization) simultaneously,followed by two boosts 4 weeks apart (days 0, 28, and 56). Twelve daysafter the initial immunization, treatment with mab 20H4.9-IgG4 or mabhu39E3.G4 was initiated. Antibodies were administered i.v, at 50 mg/kg,on days 12, 15, and 19 after the first immunization. This schedule waschosen because it was shown to suppress the antibody response to mabhu39E3.G4.

Clinical and Clinical Pathology

Throughout the course of the study, physical examinations were conductedon all monkeys by the attending veterinarians. Blood samples forhematology and serum chemistry analyses were collected prior tovaccinations and then 12, 42, 70, 97, 134, and 168 days afterimmunizations.

Immunological Assays

To determine the effect on the immune responses induced by thesetherapeutic regimens, an enzyme-linked immune spot assay (ELISPOT) wasused for the detection of IFN-γ production by antigen-specificstimulated lymphocytes. Blood samples for ELISPOT analyses werecollected prior to vaccinations and then 12, 42, 70, 97, 134, and 168days after immunizations. Synthetic peptides corresponding to thecomplete sequences of SIV gag and the PSA antigen were used for ex-vivostimulation of PBMC.

Results

Antigen-specific IFN-γ secreting cells in response to PSA or SIV gagpeptides were quantitated by ELISPOT. FIG. 16 (FIGS. 16A-16D)illustrates the results obtained from Groups 1-4, respectively. Thelevel of response to PSA was very low in all groups, indicating that thevaccine by itself did not induce a measurable and consistent immuneresponse when compared to non-vaccinated animals. On the other hand, SIVgag vaccination alone resulted in significant number of antigen-specificIFN-γ secreting cells that augmented over time (FIG. 16A). Untreatedanimals (not vaccinated) showed 100-1,000 spots/10⁶ PBMC throughout thecourse of the study (FIG. 16D). These results established the thresholdresponse to the vaccine; animals that presented <1,000 spots/10⁶ PBMCwere considered non-responders. In the group of animals that receivedvaccine, 5 out of 6 monkeys showed an increased response overtime, witha mean number of spots after the third immunization (day 70) of 1,727spots/10⁶ PBMC (SD=242, range=1,403-1,968 spots/10⁶ PBMC). One monkeywas considered a non-responder (620 spots/million PBMC). Since in thesestudies MHC typing was not done, it is likely that the lack of T cellresponses to the vaccine by some monkeys may be due to MHC-mismatch.Remarkably, on day 70, 4 out of 6 animals treated with SIV gag plus mab20H4.9-IgG4 presented a significant higher number of IFN-γ spots (FIG.16C) compared to control animals (FIG. 16D) and to macaques that wereimmunized with DNA vaccine alone (FIG. 16A). The mean number of spotsafter the third immunization for the mab 20H4.9-IgG4-treated group wasof 3,465 spots/10⁶ PBMC (SD=1,236, range=2,070-4,780 spots/10⁶ PBMC).Two monkeys in that group did not respond to the vaccine (<800spots/million PBMC). Following the third immunization (day 70),treatment with mab hu39E3.G4 plus DNA vaccine resulted in 6 out of 6animals considered as responders with a mean number of spots/10⁶ PBMC of2,348 (SD=588, range=1,738-3,283) (FIG. 16B). For this group, the rangeof the number of spots was lower compared to those macaques treated withmab 20H4.9-IgG4.

Treatment with both mab 20H4.9-IgG4 and mab hu9E3.G4 was well toleratedand did not result in any significant changes in clinical signs,clinical chemistry, or hematological parameters relative to controlmonkeys.

These data show that mab 20H4.9-IgG4 treatment in combination with a DNAvaccine elicited an in vivo enhancement of the magnitude of the specificcellular response to the test antigen relative to controls or totreatment with mab hu39E3.G4, as measured by antigen specificIFN-γ-secreting cells. Since only one dose level of the antibodies andone dosing regimen were used in these preliminary studies, it isunlikely that maximal responses were induced, and further work tooptimize conditions is required. Clearly, however, even with thisnon-optimized protocol, an enhancement of the cellular response to testantigens was achieved with mab 20H4.9-IgG4, suggesting that modulationof CD137 function may be an attractive approach for augmenting theeffectiveness of DNA vaccines.

Although the invention has been described in some detail by way ofillustration and example for purposes of clarity and understanding, itwill be apparent that certain changes and modifications may be practicedwithin the scope of the appended claims.

What is claimed is:
 1. A monoclonal antibody or antigen-binding portionthereof that specifically binds to 4-1BB, comprising a light chainvariable region and a heavy chain variable region, wherein: said lightchain variable region comprises a CDR1, a CDR2, and a CDR3 as depictedin FIG. 4; and said heavy chain variable region comprises a CDR1, aCDR2, and a CDR3 as depicted in FIG.
 3. 2. The monoclonal antibody orantigen-binding portion thereof of claim 1, wherein: said light chaincomprises a variable region as depicted in FIG. 4; and said heavy chaincomprises a variable region as depicted in FIG.
 3. 3. A monoclonalantibody comprising a light chain and a heavy chain, wherein said lightchain comprises amino acid residues 21-236 of SEQ ID NO:6 and said heavychain comprises amino acid residues 20-467 of SEQ ID NO:3.
 4. Apharmaceutical composition comprising: the monoclonal antibody orantigen-binding portion thereof of claim 1; and a pharmaceuticallyacceptable carrier.
 5. A pharmaceutical composition comprising: themonoclonal antibody of claim 3; and a pharmaceutically acceptablecarrier.
 6. A method for treating a cancer in a subject comprisingadministering a therapeutically effective amount of the monoclonalantibody or antigen-binding portion thereof of claim 1 to said subject.7. An isolated polynucleotide comprising a nucleotide sequence thatencodes the amino acid sequence of amino acid residues 20-467 of SEQ IDNO:3.
 8. The polynucleotide of claim 7 that comprises the nucleotidesequence of SEQ ID NO:1.
 9. An isolated polynucleotide comprising anucleotide sequence that encodes the amino acid sequence of amino acidresidues 21-236 of SEQ ID NO:6.
 10. The polynucleotide of claim 9 thatcomprises the nucleotide sequence of SEQ ID NO:4.